CN1205479C - 免疫学诊断蛋白芯片制备方法 - Google Patents
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Abstract
本发明公开了一种免疫学诊断蛋白芯片制备方法。蛋白芯片是在玻片、硅片、膜、高分子材料载体上固定CD1，CD2等5～200种特异性单克隆抗体及其阳性和阴性对照，它是将单克隆抗体用化学方法固定于经过戊二醛或多聚赖氨酸或小牛血清蛋白或亲和素处理过的载体上。本发明可同时检测免疫白细胞的表面抗原分型及多种蛋白表达水平。具有快速，准确，平行，多重分析特点，该芯片可用于免疫系统疾病诊断和白血病分型及蛋白表达研究。
Description
本发明涉及医学诊断，尤其涉及一种免疫学诊断蛋白芯片制备方法。
现行的生物芯片一般为基因芯片。蛋白质的固定难度大、蛋白芯片的制备工艺复杂、成本昂贵。蛋白芯片目前都是以膜为介质，如PVDF膜，NC膜，不易操作。现有的检测特异蛋白的技术，如ELISA，放射免疫方法，Westernblot，存在操作繁杂，所需时间长，同位素污染等。
本发明的目的是提供一种大规模集成化，工艺简单，准确定位，快速反应，且价格低廉，并能平行，多重分析的免疫学诊断蛋白芯片制备方法。
为了达到上述目的，本发明采取下列措施；
免疫学诊断蛋白芯片的制备方法是将0.1～10ng CD系列单克隆抗体用1～100μl的0.1～1.0M包含羟基和胺基的双官能团化合物加0.05～0.5M醋酸盐或磷酸盐混合液点样于用戊二醛或多聚赖氨酸或小牛血清蛋白或亲和素处理过的玻片、硅片、膜、高分子材料载体上，室温过夜后，用1～1 00μl的0.1～1.0M包含羟基和胺基的双官能团化合物加0.05～0.5M醋酸盐或磷酸盐混合液室温封闭0.5～4小时，用0.01～0.1M PB缓冲液冲洗干燥，0～4℃保存待用。
本发明制备的免疫学诊断蛋白芯片是在玻片、硅片、膜、高分子材料载体上用高密度阵列点样仪形成蛋白微阵列，用荧光标记的蛋白与固定在载片上的抗体发生特异结合反应，用荧光扫描方法检测免疫信号，因此可同时检测多种蛋白及蛋白表达(见图1)。具有快速，准确，平行，多重分析特点，检测时间可缩短至2小时内完成。该芯片可用于免疫系统疾病诊断和白血病分型及蛋白表达研究。
下面结合附图和实施例对本发明作详细说明。
图1是免疫学诊断蛋白芯片示意图；
图2是CD系列单克隆抗体蛋白芯片阵列图；
图3是免疫学诊断蛋白芯片结果图。
免疫学诊断蛋白芯片是在玻片、硅片、膜、高分子材料载体上固定特异性单克隆抗体，所说的特异性单克隆抗体为：CD1，CD2，CD3，CD4，CD5，CD7，CD8，CD10，CD13，CD14，CD16，CD19，CD22，CD23，CD25，CD34，CD38，CD41，CD56的特异性抗体，及阳性和阴性对照：免疫球蛋白-Kappa，免疫学诊断蛋白-Lambda，肌过氧化酶(MPO，Myeloperoxidase)，神经元特异性烯醇酶(NSE，neuron-specific enolase)，天然杀伤细胞标志物，血清细胞标志物，β-微管蛋白和小牛血清球蛋白。
实施例1：
材料与方法：
a.CD系列单克隆抗体：
CD1，CD2，CD3，CD4，CD5，CD7，CD8，CD10，CD13，CD14，CD16，CD19，CD22，CD23，CD25，CD34，CD38，CD41，CD56的特异性抗体，以及阳性和阴性对照：免疫球蛋白-Kappa，免疫学诊断蛋白-Lambda，肌过氧化酶(MPO，Myeloperoxidase)，神经元特异性烯醇酶(NSE，neuron-specificenolase)，天然杀伤细胞标志物，血清细胞标志物，β-微管蛋白和小牛血清球蛋白。
b.细胞株：
HL-60，K562，Jurkat，NB4
培养细胞的膜蛋白抽提与荧光标记：
所有培养细胞将细胞数调整到2×107，用2％TritonX-114抽提膜蛋白，用终浓度为5ug/ml_荧光素(FITC，fluorescein isothiocyante)，或终浓度为2.5ug/ml荧光偶联素(FSE，fluorescein succinimidyl ester)标记膜蛋白，标记反应在室温为一小时，用透析或离心法去除游离的荧光素。
CD系列单克隆抗体的固定：
将CD系列单克隆抗体1μl(10ng)与1μl的0.5M胺基乙醇加氰硼氢化物偶联缓冲液加0.1M醋酸钾混合液混合按一定的格式用阵列点样仪点于胺基或多聚赖胺酸处理的玻璃载片上(见图2)，40ng的BSA为阴性对照，室温过夜后，用2μl的0.5M胺基乙醇加氰硼氢化物偶联缓冲液加0.1M醋酸钾混合液室温封闭1小时，0.01M PBS缓冲液冲洗干燥，4℃保存待用。
免疫反应及信号扫描：
取10μl荧光标记的膜蛋白于CD蛋白芯片上，室温1小时，水冲洗干燥。荧光扫描，并采用软件分析图像结果。
CD系列单克隆抗体阳性为绿色荧光点，阴性无荧光点(见图3)。
实施例2
将CD系列单克隆抗体与40％甘油和PBS缓冲液混合点样于胺基修饰的玻片上反应3小时，放置于包含0.1M小牛血清蛋白溶液1小时，用PBS缓冲液清洗10分钟，室温过夜后，0～4℃保存待用。免疫反应及信号采集与实施例1相同。
实施例3
将用多聚赖胺酸修饰的玻片放置于包含0.1M小牛血清蛋白溶液1小时，用PBS缓冲液清洗10分钟，用0.5M的N，N’-disuccinimidyl carbonate处理30分钟，将CD系列单克隆抗体与40％甘油和PBS缓冲液混合点样于该玻片，室温放置过夜后，用0.01M PBS缓冲液冲洗干燥，0～4℃保存待用。免疫反应及信号采集与实施例1相同。
实施例4
将用多聚赖胺酸或胺基修饰的玻片放置于包含0.1M亲和素溶液4小时，用PBS缓冲液清洗10分钟，将用生物素标记过的CD系列单克隆抗体和40％甘油和PBS缓冲液混合点样于该玻片，室温放置过夜后，用0.01M PBS缓冲液冲洗干燥，0～4℃保存待用。免疫反应及信号采集与实施例1相同。
Claims (2)
1.一种免疫学诊断蛋白芯片的制备方法，其特征在于：将0.1～10ng CD系列单克隆抗体用1～100μl的0.1～1.0M包含羟基和胺基的双官能团化合物加0.05～0.5M醋酸盐或磷酸盐混合液点样于用戊二醛或多聚赖氨酸或小牛血清蛋白或亲和素处理过的玻片、硅片、膜、高分子材料载体上，室温过夜后，用1～100μl的0.1～1.0M包含羟基和胺基的双官能团化合物加0.05～0.5M醋酸盐或磷酸盐混合液室温封闭0.5～4小时，用0.01～0.1M PBS缓冲液冲洗干燥，0～4℃保存待用。
2.根据权利要求1所述的一种免疫学诊断蛋白芯片的制备方法，其特征在于：将CD系列单克隆抗体0.1～10ng与1～100μl的0.1～1.0M胺基乙醇加氰硼氢化物(cyanoborohydride)偶联缓冲液加0.05～0.5M醋酸钾混合液混合点样于用戊二醛或多聚赖氨酸处理过的载体上，室温过夜后，用1～100μl的0.1～1.0M胺基乙醇加氰硼氢化物偶联缓冲液加0.05～0.5M醋酸钾混合液室温封闭0.5～4小时，用0.01～0.1M PBS缓冲液冲洗干燥，0～4℃保存待用。
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| GB0210121D0 (en) * | 2002-05-02 | 2002-06-12 | Celltech R&D Ltd | Biological products |
| CN1164948C (zh) * | 2002-06-06 | 2004-09-01 | 成都夸常科技有限公司 | 一种基于抗原抗体反应的试剂盒 |
| CN1300587C (zh) * | 2004-12-20 | 2007-02-14 | 山东省医药生物技术研究中心 | 检测血液脑脊液病原体抗体的蛋白芯片及其制备方法与应用 |
| CN107033243B (zh) * | 2005-03-23 | 2020-12-15 | 根马布股份公司 | 用于治疗多发性骨髓瘤的cd38抗体 |
| CN101957378A (zh) * | 2009-07-13 | 2011-01-26 | 上海汇中细胞生物科技有限公司 | Cd4细胞芯片及其制备方法和应用 |
| CN102274762A (zh) * | 2010-06-09 | 2011-12-14 | 李彬 | 多聚赖氨酸附膜片基的制备方法 |
| CN102539740A (zh) * | 2010-12-29 | 2012-07-04 | 河北省健海生物芯片技术有限责任公司 | 可同时检测80种自身抗体的蛋白质芯片和方法 |
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| CN102786595B (zh) * | 2012-08-03 | 2014-04-23 | 无锡傲锐东源生物科技有限公司 | 抗cd5蛋白单克隆抗体及其用途 |
| CN102776154A (zh) * | 2012-08-22 | 2012-11-14 | 天津三箭生物技术有限公司 | 小鼠抗人β-Tubulin单克隆抗体及分泌该单克隆抗体的杂交瘤细胞株 |
| CN103880960B (zh) * | 2014-04-02 | 2016-01-06 | 福州迈新生物技术开发有限公司 | 一种抗cd10分子的单克隆抗体及其应用 |
| CN109060640B (zh) * | 2018-08-08 | 2021-04-06 | 上海汇中细胞生物科技有限公司 | 用于检测b淋巴细胞的cd系列细胞检测玻片 |
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